8  Microglial subpopulations

Identification of Microglial Subpopulations after reclustering

Published

August 13, 2025

8.1 Reclustered microglia

The integrated scRNAseq dataset of all brain cell populations was subsetted to retain only cells annotated as microglia by the “expert committee”. The subset was reprocessed using the assay integrated to ensure batch-corrected expression values were used for downstream clustering.

Dimensionality reduction was performed by principal component analysis (PCA) on the set of variable features. An elbow plot was generated to identify the inflection point, and the first 10 principal components were selected for neighborhood graph construction.

Nearest neighbors in the reduced space were computed and an unsupervised clustering across a range of resolutions (0.1 to 1.0, step 0.2) was performed. Cluster stability across resolutions was evaluated with the clustree package, and a resolution of 0.3 was selected as a balance between capturing subpopulation diversity and avoiding overfragmentation.

The final clusters were visualized using UMAP. The reclustering identified seven transcriptionally distinct microglial subpopulations (clusters 0–6) (Fig. 1B), compared with the initial single microglia cluster in the whole-brain dataset (Fig. 1A). These clusters exhibited clear separation in UMAP space, indicating distinct transcriptional programs.

Contribution of each original cluster (0-28) towards the reclustered clusters (0-6)
0 1 2 3 5 8 12 13 14 28
0 6007 146 101 95 0 189 14 0 3 45
1 25 4829 1 283 40 101 42 795 6 3
2 196 121 4783 3 78 45 41 0 3 65
3 47 31 1 3337 7 2 709 15 11 15
4 0 13 8 1 3397 0 0 188 6 1
5 603 18 14 17 1 2144 527 18 3 8
6 14 83 3 1 25 0 6 1 944 4

8.2 Cluster markers

Cluster-specific marker genes were determined using a Wilcoxon rank-sum test, restricted to genes expressed in at least 30% of cells within a cluster.

The table represents the top 20 markers (according to the adjusted p-value) for each cluster:

cluster top_markers
0 Nfkbia (Upregulated) , Gadd45b (Upregulated) , Tnf (Upregulated) , Egr1 (Upregulated) , Nfkbiz (Upregulated) , Ier2 (Upregulated) , Ier3 (Upregulated) , Il1b (Upregulated) , Bcl2a1d (Upregulated) , Tnfaip3 (Downregulated), Ccl4 (Upregulated) , Mir155hg (Upregulated) , Nr4a3 (Upregulated) , Junb (Upregulated) , Ifrd1 (Upregulated) , Btg2 (Downregulated) , Clic4 (Upregulated) , Fos (Downregulated) , Ccrl2 (Upregulated) , Ccl3 (Upregulated)
1 Zfp36 (Downregulated) , Btg2 (Downregulated) , Cd83 (Downregulated) , Dennd4a (Downregulated) , Atf3 (Downregulated) , Rel (Downregulated) , Ier5 (Downregulated) , Jun (Downregulated) , H3f3b (Downregulated) , Mapkapk2 (Downregulated), Baiap2l2 (Upregulated) , Cebpb (Downregulated) , Zfp36l1 (Downregulated) , Or5v1b (Upregulated) , Tmem163 (Upregulated) , Btg1 (Downregulated) , Plek (Downregulated) , Serpine2 (Upregulated) , Cst7 (Downregulated) , Ank (Downregulated)
2 Ctsb (Downregulated) , Serpine2 (Downregulated), Tgm2 (Upregulated) , Cacnb2 (Upregulated) , Klf2 (Upregulated) , Il1a (Upregulated) , Socs3 (Upregulated) , Tmem119 (Upregulated) , Trem2 (Downregulated) , Cd68 (Downregulated) , Syngr1 (Downregulated) , Fos (Upregulated) , Ccr5 (Upregulated) , Crybb1 (Upregulated) , Nr4a1 (Upregulated) , Jun (Upregulated) , Rapgef5 (Upregulated) , Ctsd (Downregulated) , Jund (Upregulated) , Rnf19b (Upregulated)
3 Spp1 (Upregulated) , Atp6v0d2 (Upregulated) , H2-Aa (Upregulated) , Gpnmb (Upregulated) , H2-Eb1 (Upregulated) , Itgax (Upregulated) , Fabp5 (Upregulated) , Dkk2 (Upregulated) , Trpc4 (Upregulated) , H2-Ab1 (Upregulated) , Ctnna3 (Upregulated) , Siglech (Downregulated), P2ry12 (Downregulated) , Cst7 (Upregulated) , Igf1 (Upregulated) , Plau (Upregulated) , Cd74 (Upregulated) , Mamdc2 (Upregulated) , Lpcat2 (Downregulated) , Lgals3 (Upregulated)
4 Cacnb2 (Upregulated) , Ctsb (Downregulated) , Plek (Downregulated) , Bank1 (Upregulated) , Gm10790 (Upregulated) , Tyrobp (Downregulated) , Frmd4a (Upregulated) , Tmem119 (Upregulated) , Ccr5 (Upregulated) , Rapgef5 (Upregulated) , Maf (Upregulated) , Cd9 (Downregulated) , Trem2 (Downregulated) , A830008E24Rik (Upregulated), Lyz2 (Downregulated) , Cd68 (Downregulated) , Ctsz (Downregulated) , Csmd3 (Upregulated) , Syngr1 (Downregulated) , Ifitm10 (Upregulated)
5 Itgam (Downregulated) , Ifi207 (Upregulated) , C1qb (Downregulated) , Ccl4 (Upregulated) , Lilrb4a (Upregulated) , Ly86 (Upregulated) , C1qc (Downregulated) , Fgf13 (Upregulated) , C1qa (Downregulated) , Klf6 (Upregulated) , Dusp1 (Upregulated) , Atf3 (Upregulated) , P2ry12 (Downregulated), Marcks (Downregulated), Rgs10 (Downregulated) , Cst3 (Downregulated) , Ctss (Downregulated) , Hexb (Downregulated) , Laptm5 (Downregulated), Xylt1 (Upregulated)
6 Ifit3 (Upregulated) , Ifi211 (Upregulated) , Ifi206 (Upregulated) , Ifit2 (Upregulated) , Rsad2 (Upregulated) , Isg15 (Upregulated) , Mx1 (Upregulated) , Ifit3b (Upregulated) , Ifi213 (Upregulated) , Oasl2 (Upregulated) , A330040F15Rik (Upregulated), Ifi209 (Upregulated) , Slfn5 (Upregulated) , Gm4951 (Upregulated) , Ifitm3 (Upregulated) , Stat1 (Upregulated) , Ifi207 (Upregulated) , Rnf213 (Upregulated) , Sp100 (Upregulated) , Herc6 (Upregulated)

8.3 Cluster interpretation (chatGPT 5)

8.3.1 Cluster 0

Gene Cell Subtype Regulation Notes
Nfkbia Microglia Activated/inflammatory microglia (AIM) Upregulated NF-κB inhibitor; rapid-response gene in inflammatory microglia during cytokine signaling.
Gadd45b Microglia Activated/inflammatory microglia (AIM) Upregulated Stress-responsive transcriptional regulator induced by pro-inflammatory stimuli in microglia.
Tnf Microglia Activated/inflammatory microglia (AIM) Upregulated Classic pro-inflammatory cytokine, hallmark of microglial activation.
Egr1 Microglia Immediate early gene (IEG)-activated microglia Upregulated Rapid transcriptional activator in microglia after acute stimuli; linked to inflammatory and stress responses.
Nfkbiz Microglia Activated/inflammatory microglia (AIM) Upregulated NF-κB pathway regulator; modulates pro-inflammatory gene expression in microglia.
Ier2 Microglia Immediate early gene (IEG)-activated microglia Upregulated Early-response transcription factor upregulated in activated microglia after stimulation.
Ier3 Microglia Immediate early gene (IEG)-activated microglia Upregulated Anti-apoptotic and stress response mediator; induced in activated microglia.
Il1b Microglia Activated/inflammatory microglia (AIM) Upregulated Key pro-inflammatory cytokine secreted by activated microglia; promotes neuroinflammation.
Bcl2a1d Microglia Activated/inflammatory microglia (AIM) Upregulated Anti-apoptotic protein induced by NF-κB in inflammatory microglia.
Tnfaip3 Microglia Activated/inflammatory microglia (AIM) Downregulated Negative regulator of NF-κB signaling; lower levels can prolong inflammatory responses.
Ccl4 Microglia Chemotactic/inflammatory microglia Upregulated Chemokine attracting immune cells; enriched in DAM/AIM microglia.
Mir155hg Microglia Activated/inflammatory microglia (AIM) Upregulated Host gene for miR-155, a pro-inflammatory regulator in microglia.
Nr4a3 Microglia Immediate early gene (IEG)-activated microglia Upregulated Nuclear receptor involved in transcriptional reprogramming after activation.
Junb Microglia Immediate early gene (IEG)-activated microglia Upregulated Component of AP-1 transcription factor complex; induced by inflammatory and stress signals.
Ifrd1 Microglia Activated/inflammatory microglia (AIM) Upregulated Transcriptional regulator linked to immune cell activation.
Btg2 Microglia Immediate early gene (IEG)-activated microglia Downregulated Early-response gene; reduction may reflect shift from transient to sustained activation.
Clic4 Microglia Activated/inflammatory microglia (AIM) Upregulated Chloride channel involved in microglial activation and phagocytosis.
Fos Microglia Immediate early gene (IEG)-activated microglia Downregulated Classic IEG; lower levels may indicate post-acute or chronic activation phase.
Ccrl2 Microglia Chemotactic/inflammatory microglia Upregulated Chemokine receptor-like protein promoting immune cell recruitment during neuroinflammation.
Ccl3 Microglia Chemotactic/inflammatory microglia Upregulated Pro-inflammatory chemokine; signature of activated and DAM microglia.

All 20 genes are microglial, with the majority linked to activated/inflammatory microglia (AIM) and chemotactic responses, indicating strong pro-inflammatory activation. A large subset are immediate early genes (Egr1, Ier2, Ier3, Nr4a3, Junb, Fos, Btg2), suggesting acute transcriptional reprogramming. Several chemokines (Ccl3, Ccl4) and receptors (Ccrl2) point to immune cell recruitment, while NF-κB pathway components (Nfkbia, Nfkbiz, Tnfaip3) highlight central inflammatory signaling. The pattern is consistent with a robust, possibly sustained inflammatory microglial state with recruitment and signaling functions active.

8.3.2 Cluster 1

Gene Cell Subtype Regulation Notes
Zfp36 Microglia Homeostatic microglia Downregulated RNA-binding protein promoting resolution of inflammation; reduced expression suggests loss of anti-inflammatory tone.
Btg2 Microglia Immediate early gene (IEG)-activated microglia Downregulated Early-response regulator; decreased levels may indicate reduced acute transcriptional activation.
Cd83 Microglia Activated/antigen-presenting microglia Downregulated Co-stimulatory molecule; lower levels suggest dampened antigen presentation capacity.
Dennd4a Microglia Homeostatic microglia Downregulated Endosomal trafficking regulator; reduced expression may impair normal vesicular function.
Atf3 Microglia Immediate early gene (IEG)-activated microglia Downregulated Stress-responsive transcription factor; decrease suggests reduced acute stress response.
Rel Microglia Activated/inflammatory microglia (AIM) Downregulated NF-κB subunit; downregulation indicates suppressed inflammatory transcription program.
Ier5 Microglia Immediate early gene (IEG)-activated microglia Downregulated Early-response transcriptional regulator; lower levels indicate reduced activation.
Jun Microglia Immediate early gene (IEG)-activated microglia Downregulated AP-1 complex member; decreased expression points to reduced stimulus-driven gene expression.
H3f3b Microglia Homeostatic microglia Downregulated Histone variant linked to active transcription; reduction may reflect lower transcriptional turnover.
Mapkapk2 Microglia Activated/inflammatory microglia (AIM) Downregulated p38 MAPK pathway kinase; downregulation suggests decreased cytokine production signaling.
Baiap2l2 Microglia Potentially reactive microglia Upregulated Actin cytoskeleton regulator; increased levels may support morphological remodeling.
Cebpb Microglia Activated/inflammatory microglia (AIM) Downregulated Transcription factor driving inflammatory and DAM programs; reduced levels suggest attenuated activation.
Zfp36l1 Microglia Homeostatic microglia Downregulated Post-transcriptional regulator of inflammatory mRNAs; decrease may affect RNA stability control.
Or5v1b Microglia Uncharacterized/possible off-target expression Upregulated Olfactory receptor-like; rare in microglia, upregulation could reflect atypical activation.
Tmem163 Microglia Homeostatic microglia Upregulated Vesicular zinc transporter; increase may relate to altered ion handling in reactive states.
Btg1 Microglia Immediate early gene (IEG)-activated microglia Downregulated Cell cycle regulator with early-response profile; reduction aligns with quiescence.
Plek Microglia Homeostatic microglia Downregulated Cytoskeletal-associated protein; decrease may reflect reduced motility or phagocytic activity.
Serpine2 Microglia Remodeling/reactive microglia Upregulated Extracellular matrix regulator; elevated in tissue remodeling and repair contexts.
Cst7 Microglia Activated/DAM microglia Downregulated Cysteine protease inhibitor; lower expression may impair control of proteolytic activity.
Ank Microglia Homeostatic microglia Downregulated Cytoskeletal anchoring protein; decrease may indicate reduced stability of cell processes.

Most genes in cluster 1 are downregulated and associated with homeostatic, immediate early, or inflammatory microglial programs, indicating an overall suppression of activation and immune signaling. Notably, transcription factors (Rel, Jun, Cebpb) and RNA regulators (Zfp36, Zfp36l1) are decreased, suggesting reduced inflammatory transcription and mRNA turnover. A few genes (Baiap2l2, Tmem163, Serpine2) are upregulated, hinting at selective morphological remodeling and extracellular matrix modulation. Overall, the profile suggests a shift toward a less inflammatory, potentially partially remodeling/reactive microglial state.

8.3.3 Cluster 2

Gene Cell Subtype Regulation Notes
Ctsb Microglia Lysosomal/phagocytic (DAM-like) Downregulated Cathepsin B; reduced levels suggest diminished phagolysosomal activity typical of DAM/phagocytic programs.
Serpine2 Microglia Remodeling/reactive Downregulated ECM serine protease inhibitor; decrease implies less extracellular remodeling.
Tgm2 Microglia Activated/reactive microglia Upregulated Transglutaminase 2; induced in activated microglia, supports adhesion/phagocytosis and Aβ handling.
Cacnb2 Microglia Reactive signaling microglia Upregulated Ca²⁺ channel β subunit; heightened Ca²⁺ signaling aligns with reactive state transitions.
Klf2 Microglia Homeostatic/anti‑inflammatory Upregulated Transcription factor that restrains inflammatory programs; rise suggests homeostatic pushback.
Il1a Microglia Activated/inflammatory microglia (AIM) Upregulated Pro‑inflammatory cytokine driving local neuroinflammation.
Socs3 Microglia Activated (JAK/STAT feedback) Upregulated Negative feedback regulator of cytokine signaling; elevation marks acute activation with braking.
Tmem119 Microglia Homeostatic microglia Upregulated Core microglial identity marker; increase indicates preserved homeostatic signature.
Trem2 Microglia DAM/phagocytic Downregulated Key receptor for DAM induction and lipid/Aβ phagocytosis; drop argues against DAM engagement.
Cd68 Microglia Phagocytic/activated Downregulated Lysosomal/phagolysosomal marker; reduction indicates lower phagocytic tone.
Syngr1 Microglia Contamination/neuronal signal Downregulated Synaptic vesicle protein (neuronal); decreased expression fits reduced ambient neuronal RNA.
Fos Microglia Immediate‑early gene (IEG)‑activated Upregulated Classic IEG; marks acute transcriptional activation.
Ccr5 Microglia Chemotactic/inflammatory Upregulated Chemokine receptor; suggests migration/recruitment during inflammatory response.
Crybb1 Microglia Stress‑responsive/reactive Upregulated Crystallin family chaperone; induced under cellular stress in glia.
Nr4a1 Microglia IEG/anti‑inflammatory reprogramming Upregulated Nuclear receptor shaping rapid activation and tolerance programs.
Jun Microglia IEG‑activated Upregulated AP‑1 component; hallmark of acute stimulus response.
Rapgef5 Microglia Motility/chemotaxis signaling Upregulated Rap1 GEF; supports cytoskeletal dynamics and process motility in reactive microglia.
Ctsd Microglia Lysosomal/phagocytic (DAM-like) Downregulated Cathepsin D; decrease reinforces reduced lysosomal/phagocytic program.
Jund Microglia IEG‑activated Upregulated AP‑1 family factor; marks early activation.
Rnf19b Microglia Stress/adaptive (proteostasis) Upregulated E3 ubiquitin ligase (ERAD/quality control); elevation indicates proteostatic stress response.

Cluster 2 combines strong IEG/cytokine activation (Fos/Jun/Jund, Il1a, Socs3, Ccr5) with preservation of homeostatic identity (Tmem119, Klf2↑) and suppression of DAM/phagolysosomal genes (Trem2, Cd68, Ctsb, Ctsd↓). This pattern supports an acute, non‑phagocytic inflammatory microglial state with chemotactic/migratory bias and active negative‑feedback (Socs3), rather than a classic Trem2⁺ DAM phenotype. Tgm2 and stress markers (Crybb1, Rnf19b) suggest matrix/adhesion changes and proteostatic adaptation during this transient activation.

8.3.4 Cluster 3:

Gene Cell Subtype Regulation Notes
Spp1 Microglia Spp1⁺ DAM (osteopontin-high) Upregulated Canonical Spp1⁺ neurodegeneration-associated subset; linked to phagolysosomal activation and plaque-proximal microglia.
Atp6v0d2 Microglia Spp1⁺ DAM Upregulated Lysosomal/osteoclast-like proton pump subunit enriched in Spp1⁺ DAM; supports catabolic/phagolysosomal programs.
H2-Aa Microglia MHC-II⁺ antigen-presenting microglia (ARM) Upregulated MHC-II component indicating antigen presentation.
Gpnmb Microglia DAM/MGnD Upregulated Injury/DAM marker enriched in neurodegenerative microglia and lipid-handling states.
H2-Eb1 Microglia MHC-II⁺ ARM Upregulated MHC-II beta-chain; antigen presentation program.
Itgax Microglia DAM/MGnD (CD11c⁺) Upregulated CD11c⁺ “activated”/MGnD microglia; plaque-associated.
Fabp5 Microglia Lipid-handling DAM / LDAM-like Upregulated Fatty-acid–binding protein; lipid metabolism and phagocytosis in activated microglia.
Dkk2 Microglia Neurodegenerative-DAM subset Upregulated Wnt antagonist selectively induced in mouse neurodegenerative microglia near plaques.
Trpc4 Microglia Activated/DAM-associated Upregulated Ca²⁺ channel up in activated microglia; supports motility/chemotaxis in DAM contexts.
H2-Ab1 Microglia MHC-II⁺ ARM Upregulated MHC-II alpha-chain; part of antigen-presenting signature.
Ctnna3 Microglia Activated/DAM-associated Upregulated Cell-adhesion gene reported in activated/DAM-like microglia; may reflect contact with dystrophic neurites.
Siglech Microglia Homeostatic Downregulated Homeostatic microglia marker; its loss marks transition to non-homeostatic/DAM states.
P2ry12 Microglia Homeostatic Downregulated Core homeostatic microglia receptor; down in activated/DAM microglia.
Cst7 Microglia DAM/MGnD Upregulated Cathepsin inhibitor robustly induced in DAM; linked to lysosomal remodeling.
Igf1 Microglia IGF1⁺ reparative microglia Upregulated Trophic/reparative program; promotes oligodendrocyte/myelin support within disease context.
Plau Microglia Activated ECM-remodeling/DAM Upregulated uPA; extracellular matrix remodeling and migration; increased in activated plaque-associated microglia.
Cd74 Microglia MHC-II⁺ ARM Upregulated Invariant chain for MHC-II; hallmark of antigen-presenting microglia.
Mamdc2 Microglia Activated/DAM-associated Upregulated Extracellular/adhesion protein reported in activated plaque-proximal microglia subsets.
Lpcat2 Microglia Pro‑inflammatory eicosanoid program Downregulated Arachidonoyl-lysophosphatidylcholine acyltransferase; lower levels suggest shift away from classical lipid‑inflammatory signaling.
Lgals3 Microglia Activated/DAM (Galectin‑3⁺) Upregulated Galectin‑3 drives phagocytosis and TREM2 signaling; strong DAM/plaqued‑associated marker.

Most markers map to disease-associated microglia (DAM), including a prominent Spp1⁺/osteoclast‑like module (Spp1, Atp6v0d2, Gpnmb, Fabp5, Dkk2, Itgax, Cst7, Lgals3) and an antigen-presenting MHC‑II arm (H2-Aa/H2-Ab1/H2-Eb1, Cd74). Homeostatic signatures (P2ry12, Siglech) are down, consistent with an activated, plaque‑proximal state. Additional genes (Plau, Trpc4, Ctnna3, Mamdc2) support activation/motility and ECM remodeling, while Igf1 suggests a concurrent reparative program. Overall, cluster 3 reflects plaque‑associated, antigen‑presenting Spp1⁺ DAM with suppressed homeostatic identity.

8.3.5 Cluster 4

Gene Cell Subtype Regulation Notes
Cacnb2 Microglia Reactive signaling microglia Upregulated Voltage-gated Ca²⁺ channel β subunit; promotes altered Ca²⁺ dynamics during activation and migration.
Ctsb Microglia Lysosomal/phagocytic (DAM-like) Downregulated Cathepsin B; decrease suggests reduced lysosomal proteolysis and phagocytic capacity.
Plek Microglia Homeostatic microglia Downregulated Cytoskeletal-associated protein; reduced levels may impair motility and process extension.
Bank1 Microglia Immune signaling–biased microglia Upregulated Scaffold protein in B/TLR signaling; induction suggests immune receptor pathway engagement.
Gm10790 Microglia Uncharacterized/reactive Upregulated Predicted gene with reported glial expression in activation contexts.
Tyrobp Microglia DAM/phagocytic Downregulated Adaptor for TREM2/Syk signaling; lower levels indicate reduced DAM engagement.
Frmd4a Microglia Reactive/motility remodeling Upregulated Actin cytoskeleton regulator; supports polarity and migration.
Tmem119 Microglia Homeostatic microglia Upregulated Core homeostatic marker; maintenance suggests preserved identity amid activation.
Ccr5 Microglia Chemotactic/inflammatory Upregulated Chemokine receptor driving migration toward inflammatory cues.
Rapgef5 Microglia Motility/chemotaxis signaling Upregulated Regulates Rap1 activity and cytoskeletal rearrangement in reactive microglia.
Maf Microglia Anti-inflammatory/regulatory Upregulated Transcription factor promoting repair/tolerogenic states; elevated in certain homeostatic-like contexts.
Cd9 Microglia DAM/phagocytic Downregulated Tetraspanin in exosome/lysosome biology; decrease indicates reduced vesicular/phagocytic functions.
Trem2 Microglia DAM/phagocytic Downregulated Receptor essential for DAM induction; lower expression suggests non-DAM inflammatory state.
A830008E24Rik Microglia Uncharacterized/reactive Upregulated Predicted transcript enriched in activated glia.
Lyz2 Microglia DAM/phagocytic Downregulated Lysozyme C2; loss indicates reduced microbicidal/lysosomal activity.
Cd68 Microglia Lysosomal/phagocytic Downregulated Lysosomal membrane glycoprotein; decline suggests reduced phagolysosomal activity.
Ctsz Microglia Lysosomal/phagocytic Downregulated Cathepsin Z; decrease aligns with downregulated phagolysosomal program.
Csmd3 Microglia Reactive/adhesion-modulating Upregulated Large complement regulatory protein; linked to cell–cell/ECM interactions.
Syngr1 Microglia Contamination/neuronal signal Downregulated Synaptic vesicle protein; likely ambient neuronal transcript reduced in this cluster.
Ifitm10 Microglia Stress-adaptive Upregulated IFITM family member; up in stress/injury contexts, potential role in membrane remodeling.

Cluster 4 shows a mixed profile: upregulation of signaling, motility, and immune-modulating genes (Cacnb2, Bank1, Ccr5, Rapgef5, Maf) with concurrent preservation of homeostatic identity (Tmem119↑) and marked downregulation of DAM/phagolysosomal machinery (Trem2, Tyrobp, Cd68, Ctsb, Ctsz, Lyz2). This suggests a non-DAM reactive state biased toward chemotaxis and cytoskeletal remodeling rather than debris clearance. The pattern could reflect microglia mobilized for migration or vascular surveillance, rather than plaque-engulfment, potentially relevant to vascular and inflammatory components of ARIA where microglial motility and cytokine signaling are heightened while phagocytosis is reduced.

8.3.6 Cluster 5

Gene Cell Subtype Regulation Notes
Itgam Microglia DAM/phagocytic Downregulated Encodes CD11b; key for adhesion and phagocytosis; reduction indicates lowered phagocytic activation.
Ifi207 Microglia Interferon-responsive/reactive Upregulated IFN-inducible gene; upregulation marks antiviral/innate immune activation.
C1qb Microglia Complement-mediated DAM Downregulated Complement component; downregulation suggests reduced synaptic pruning and opsonization.
Ccl4 Microglia Chemotactic/inflammatory Upregulated Pro-inflammatory chemokine; promotes immune cell recruitment to CNS lesions.
Lilrb4a Microglia Regulatory/immune-checkpoint Upregulated Inhibitory receptor; induced in inflammatory contexts to limit activation.
Ly86 Microglia Innate immune/TLR signaling Upregulated MD-1 protein; modulates TLR4/MD-2 signaling in microglial innate immune responses.
C1qc Microglia Complement-mediated DAM Downregulated Complement cascade subunit; loss further supports reduced opsonization activity.
Fgf13 Microglia Reactive/stress-adaptive Upregulated Cytosolic growth factor family member; linked to microtubule stabilization in reactive states.
C1qa Microglia Complement-mediated DAM Downregulated Initiator of classical complement pathway; decreased expression suggests suppressed complement activity.
Klf6 Microglia Pro-inflammatory transcriptional reprogramming Upregulated Induced by stress/injury; regulates inflammatory and migratory genes.
Dusp1 Microglia Anti-inflammatory/feedback Upregulated MAPK phosphatase; limits MAPK-driven pro-inflammatory signaling.
Atf3 Microglia Immediate-early gene (IEG)-activated Upregulated Stress-responsive transcription factor; marks acute activation.
P2ry12 Microglia Homeostatic Downregulated Purinergic receptor; loss is a hallmark of activated, non-homeostatic states.
Marcks Microglia Cytoskeletal/homeostatic Downregulated Membrane–actin crosslinker; reduced levels impair process motility and membrane dynamics.
Rgs10 Microglia Homeostatic/anti-inflammatory Downregulated Regulator of G-protein signaling; decreased levels permit stronger chemokine receptor signaling.
Cst3 Microglia Homeostatic/lysosomal Downregulated Cystatin C; lysosomal protease inhibitor; loss suggests altered proteolytic control.
Ctss Microglia DAM/phagocytic Downregulated Cathepsin S; key for antigen processing and myelin degradation; reduced expression indicates diminished phagolysosomal activity.
Hexb Microglia Homeostatic Downregulated β-Hexosaminidase subunit; essential for lysosomal function; decrease reflects impaired lysosomal metabolism.
Laptm5 Microglia Lysosomal/immune activation Downregulated Lysosomal membrane protein; reduction suggests suppressed lysosomal activity.
Xylt1 Microglia ECM remodeling/reactive Upregulated Glycosyltransferase in proteoglycan biosynthesis; may reflect ECM reorganization during injury response.

Cluster 5 exhibits a marked downregulation of complement components (C1qa/b/c) and multiple lysosomal/phagocytic markers (Itgam, Ctss, Hexb, Laptm5), indicating a suppressed DAM-like phagocytic program. Concurrently, upregulated genes point to a mixed interferon-responsive and inflammatory signaling state (Ifi207, Ccl4, Ly86, Klf6, Atf3) with regulatory feedback mechanisms (Dusp1, Lilrb4a) and ECM remodeling (Xylt1). Loss of homeostatic markers (P2ry12, Marcks, Rgs10, Cst3) reinforces a non-homeostatic activated phenotype, biased toward cytokine production and immune modulation rather than debris clearance.

8.3.7 Cluster 6

Gene Cell Subtype Regulation Notes
Ifit3 Microglia Type I interferon–responsive microglia (IRMs) Upregulated IFN-induced protein; hallmark of antiviral/innate immune activation in microglia.
Ifi211 Microglia IRMs Upregulated Interferon-inducible family member; part of broad IFN-stimulated gene (ISG) program.
Ifi206 Microglia IRMs Upregulated ISG; contributes to innate antiviral defense.
Ifit2 Microglia IRMs Upregulated Antiviral effector limiting viral replication; robustly induced by IFN signaling.
Rsad2 Microglia IRMs Upregulated Viperin; antiviral protein regulating lipid metabolism during innate responses.
Isg15 Microglia IRMs Upregulated Ubiquitin-like modifier; critical in IFN-induced protein modification during antiviral states.
Mx1 Microglia IRMs Upregulated Dynamin-like GTPase; classical ISG conferring antiviral resistance.
Ifit3b Microglia IRMs Upregulated Paralog of Ifit3; IFN-inducible, part of viral RNA sensing and translation suppression pathway.
Ifi213 Microglia IRMs Upregulated ISG with poorly characterized function; linked to innate immunity.
Oasl2 Microglia IRMs Upregulated 2′–5′-oligoadenylate synthase–like; antiviral RNA degradation pathway.
A330040F15Rik Microglia IRMs Upregulated Predicted IFN-inducible transcript; uncharacterized in function.
Ifi209 Microglia IRMs Upregulated Member of IFN-inducible family; potential nucleic acid sensor.
Slfn5 Microglia IRMs Upregulated Schlafen family protein; modulates immune responses and cell cycle during activation.
Gm4951 Microglia IRMs Upregulated Predicted IFN-inducible gene; enriched in viral defense transcriptional programs.
Ifitm3 Microglia IRMs Upregulated Restricts viral entry by modifying endosomal membranes.
Stat1 Microglia IRMs Upregulated Master transcription factor for type I/II IFN responses; drives IRM transcriptional network.
Ifi207 Microglia IRMs Upregulated ISG; part of IFN-driven antiviral gene set.
Rnf213 Microglia IRMs Upregulated E3 ligase with roles in immunity and vascular remodeling; induced by IFNs.
Sp100 Microglia IRMs Upregulated Nuclear body protein; upregulated in antiviral and inflammatory contexts.
Herc6 Microglia IRMs Upregulated E3 ligase mediating ISGylation; essential for ISG15 conjugation pathway.

Cluster 6 shows uniform upregulation of type I interferon–stimulated genes (ISGs), including antiviral effectors (Ifit3, Rsad2, Mx1, Ifitm3), nucleic acid sensors (Oasl2, Stat1, Sp100), and ISGylation machinery (Isg15, Herc6). This profile is characteristic of interferon-responsive microglia (IRMs), a distinct activation state often triggered by viral infection, nucleic acid release from damaged cells, or chronic inflammatory cues. The absence of downregulated homeostatic or phagocytic genes suggests a strong, focused IRM polarization rather than mixed activation with DAM or AIM features.

8.3.8 Summary

Cluster Literature-defined subtype Key evidence from markers Canonical refs
0 Reactive/inflammatory microglia (NF‑κB/IEG‑high AIM) Cytokines/chemokines and NF‑κB/IEG axis (Tnf, Il1b, Ccl3/4, Nfkbia, Nfkbiz, Egr1, Junb) indicating an acute inflammatory transcriptional program distinct from DAM. Frameworks distinguishing reactive vs homeostatic/DAM microglia and nomenclature guidance. (ScienceDirect, PMC)
1 Transitional/homeostatic‑leaning (resolution/quiescent) Broad downregulation of IEGs and inflammatory TFs (Jun, Rel, Atf3, Zfp36 family) with minimal activation markers → consistent with a resolving, low‑reactivity state along the reactive→homeostatic continuum. Microglial state continuum and resolution concepts. (ScienceDirect, Frontiers)
2 Reactive/IEG‑activated (AIM) with preserved identity IEG/cytokine program (Fos, Jun, Il1a, Socs3, Ccr5) but retention of homeostatic identity (Tmem119↑, Klf2↑) and non‑DAM features (Trem2/Cd68/Ctsb/Ctsd↓). Distinction of reactive/“activated response” states from DAM and homeostatic cores. (Cell, PMC)
3 Spp1⁺ DAM with MHC‑II⁺ antigen‑presenting ARM module Canonical DAM/MGnD markers (Spp1, Gpnmb, Itgax, Cst7, Lgals3) with suppression of homeostatic genes (P2ry12↓), plus strong MHC‑II antigen‑presentation (H2‑Aa/Ab1/Eb1, Cd74). DAM/MGnD (TREM2–APOE axis) and ARM/antigen‑presenting programs. (ScienceDirect, PMC, Cell)
4 Chemotactic/migratory reactive microglia (non‑DAM) Motility/chemotaxis signaling (Ccr5, Rapgef5, Cacnb2, Frmd4a) retained identity (Tmem119↑) with phagolysosomal/DAM axis down (Trem2, Tyrobp, Cd68, Ctsb/Ctsz, Lyz2↓) → reactive but non‑phagocytic. Reactive (non‑DAM) states within current nomenclature; distinctions from DAM signature. (ScienceDirect, Cell)
5 Cytokine/immune‑modulatory reactive microglia with complement/lysosome‑low IFN‑tinted inflammatory program (Ifi207, Atf3, Klf6, Ccl4 ↑) alongside reduced complement/lysosomal/DAM machinery (C1qa/b/c, Ctss, Hexb, Laptm5 ↓) and loss of homeostatic markers (P2ry12↓). Reviews summarizing non‑DAM reactive states vs DAM/complement modules. (ScienceDirect, Alzheimer’s Journals)
6 Interferon‑responsive microglia (IRM) Uniform ISG induction (Ifit2/3/3b, Isg15, Mx1, Rsad2, Oasl2, Stat1, Herc6) defining the IFN‑I program in microglia. IRM definition and markers in mouse brain. (PMC, Cell)

Across clusters, we observe classic Spp1⁺ DAM/ARM features (cluster 3), a robust IFN‑I IRM state (cluster 6), and multiple reactive, non‑DAM states (clusters 0, 2, 4, 5) differing by IEG/NF‑κB intensity, chemotaxis/motility bias, and complement/lysosome suppression. One cluster trends toward resolution/quiescence (cluster 1). Together, these assignments align with established microglial taxonomies distinguishing homeostatic, reactive (AIM/ARM), DAM/MGnD, and IRM states in mouse models of neurodegeneration and inflammation. (ScienceDirect, PMC, Cell)